While islet transplantation shows promise as a long-term treatment for Type 1 diabetes, the supply of available donor tissue is limiting. It is thus critical to identify factors that regulate islet differentiation and function, with the goal of generating a replenishable supply of islets from pancreatic or embryonic stem cells. The HNF6 transcription factor is critical for endocrine differentiation and islet function. HNF6 is expressed throughout the pancreas during development, but becomes permanently down-regulated in endocrine cells just before birth, as islets are forming. HNF6 -/- mice have delayed endocrine differentiation, fewer endocrine cells, abnormal islet architecture, and diabetes. HNF6 activated expression of the critical proendocrine transcription factor, ngn3 (ngn3 mutant mice lack all pancreatic endocrine cells). The results of the global HNF6 knockout demonstrate that HNF6 function is essential for normal endocrine development, but can not distinguish whether HNF6 is required only at the earliest stages of endocrine development to activate ngn3, or whether HNF6 functions continuously throughout endocrine development to promote terminal differentiation of endocrine cells. The goal of this proposal is to elucidate the role of HNF6 during islet endocrine development. Using mice in which HNF6 can be either up-regulated or inactivated conditionally, we will analyze the role of HNF6 in specification, proliferation, and differentiation of pancreatic cells to the endocrine lineage. To dissect the role of HNF6 in endocrine precursor proliferation and differentiation, HNF6 expression will be activated at different developmental time points, using a reversibly inducible endocrine-specific transgene. We will determine whether HNF6 over-expression during early embryonic stages stimulates increased specification and/or differentiation of endocrine precursors and the developmental window during which HNF6 can affect endocrine specification. Using cell type-specific and inducible Cre-lox strategies, the HNF6 gene will be selectively inactivated at different stages along the pathway of endocrine development including: progenitor specification, proliferation, diversification of endocrine cell types, and terminal differentiation. These studies will determine the embryonic stage(s) during which HNF6 plays a critical role in the development of the endocrine lineage, i.e. is HNF6 needed only at early stages of endocrine specification, or is it required continuously during embryogenesis for terminal differentiation and acquisition of mature function.